(d) R1 is (X-(Z)-)n[(C3–C10)cycloalkyl]-(Z′)- wherein Z and Z′ are individually (C1–C6)alkyl, optionally interrupted by 1–3 S or non-peroxide O, or is absent, and n is 1–3; or a pharmaceutically acceptable salt thereof;

in combination with a carrier; wherein the composition is suitable for oral administration.

2. The composition of claim 1 wherein 5′-X is —CH2OH or —C(O)NR3R4.

3. The composition of claim 2 wherein 5′-X is —C(O)NR3R4.

4. The composition of claim 2 wherein R3 is H and R4 is (C1–C4)alkyl.

5. The composition of claim 1 wherein each R is H or (C1–C4)alkyl.

6. The composition of claim 1 wherein Z′ is —CH2— or —CH2—CH2—.

7. The composition of claim 6 wherein Z is —CH2— or —CH2—CH2—.

8. The composition of claim 1 wherein C3–C10 cycloalkyl group of R1 is cyclohexyl or cyclopentyl.

9. The composition of claim 8 wherein X is (C1–C4)alkoxycarbonyl, —C(O)NR3R4 or acetoxymethyl.

10. The composition of claim 8 wherein X is carboxy.

11. The composition of claim 8 wherein X-Z and Z′ are trans.

12. The composition of claim 1 wherein R is H, X is ethylaminocarbonyl, and R1 is 2-(4-methoxycarbonylcyclohexylmethyl).

13. The composition of claim 1 wherein R is H, X is ethylaminocarbonyl, and R1 is 2-(4-acetoxymethylcyclohexylmethyl).

14. The composition of claim 1 wherein the compound is methyl 4-(3-{9-6-aminopurin-2-yl)}prop-2-ynyl)cyclohexane-carboxylate.

15. The composition of claim 1 further comprising a Type IV phosphodiesterase inhibitor.

16. The composition of claim 15 wherein the inhibitor is rolipram.

17. The composition of claim 1 wherein the composition is a powder, a gelatin capsule, or compressed into a tablet.

The present invention was made with the assistance of U.S. Government funding (NIH Grant ROL HL37942). The U.S. Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for preventing tissue injury, i.e., due to inflammatory activity.

BACKGROUND OF THE INVENTION

The inflammatory response serves the purpose of eliminating harmful agents from the body. There is a wide range of pathogenic insults that can initiate an inflammatory response including infection, allergens, autoimmune stimuli, immune response to transplanted tissue, noxious chemicals, and toxins, ischemia/reperfusion, hypoxia, mechanical and thermal trauma. Inflammation normally is a very localized action which serves in expulsion, attenuation by dilution, and isolation of the damaging agent and injured tissue. The body's response becomes an agent of disease when it results in inappropriate injury to host tissues in the process of eliminating the targeted agent, or responding to a traumatic insult.

The release of inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) by leukocytes is a means by which the immune system combats pathogenic invasions, including infections. TNFα stimulates the expression and activation of adherence factors on leukocytes and endothelial cells, primes neutrophils for an enhanced inflammatory response to secondary stimuli and enhances adherent neutrophil oxidative activity. See, Sharma et al., cited above. In addition, macrophages/dendritic cells act as accessory cells processing antigen for presentation to lymphocytes. The lymphocytes, in turn, become stimulated to act as pro-inflammatory cytotoxic cells.

Generally, cytokines stimulate neutrophils to enhance oxidative (e.g., superoxide and secondary products) and nonoxidative (e.g., myeloperoxidase and other enzymes) inflammatory activity. Inappropriate and over-release of cytokines can produce counterproductive exaggerated pathogenic effects through the release of tissue-damaging oxidative and nonoxidative products (K. G. Tracey et al., J. Exp. Med., 167, 1211 (1988); and D. N. Männel et al., Rev. Infect. Dis., 9 (suppl. 5), S602–S606 (1987)). For example, TNFα can induce neutrophils to adhere to the blood vessel wall and then to migrate through the vessel to the site of injury and release their oxidative and non-oxidative inflammatory products.

It has been suggested that there is more than one subtype of adenosine receptor on neutrophils that can have opposite effects on superoxide release (B. N. Cronstein et al., J. Clin. Invest., 85, 1150 (1990)). The existence of A2A receptor on neutrophils was originally demonstrated by Van Calker et al. (D. Van Calker et al., Eur. J. Pharmacology, 206, 285 (1991)).

There has been progressive development of compounds that are more and more potent and/or selective as agonists of A2A adenosine receptors (AR) based on radioligand binding assays and physiological responses. Initially, compounds with little or no selectivity for A2A receptors were developed, such as adenosine itself or 5′-carboxamides of adenosine, such as 5′-N-ethylcarboxamidoadenosine (NECA) (B. N. Cronstein et al., J. Immunol., 135, 1366 (1985)). Later, it was shown that addition of 2-alkylamino substituents increased potency and selectivity, e.g., CV1808 and CGS21680 (M. F. Jarvis et al., J. Pharmacol. Exp. Ther., 251, 888 (1989)). 2-Alkoxy-substituted adenosine derivatives such as WRC-0090 are even more potent and selective as agonists at the coronary artery A2A receptor (M. Ueeda et al., J. Med. Chem., 34, 1334 (1991)). The 2-alklylhydrazino adenosine derivatives, e.g., SHA 211 (also called WRC-0474) have also been evaluated as agonists at the coronary artery A2A receptor (K. Niiya et al., J. Med. Chem., 35, 4557 (1992)).

There is one report of the combination of relatively nonspecific adenosine analogs, R-phenylisopropyladenosine (R-PIA) and 2-chloroadenosine (Cl-Ado) with a phosphodiesterase (PDE) inhibitor resulting in a lowering of neutrophil oxidative activity (M. A. Iannone et al., Topics and Perspectives in Adenosine Research, E. Garlach et al., eds., Springer-Verlag, Berlin, pp. 286–298 (1987)). However, R-PIA and Cl-Ado analogs are actually more potent activators of A1 adenosine receptors than of A2A adenosine receptors and, thus, are likely to cause side effects due to activation of A1 receptors on cardiac muscle and other tissues causing effects such as “heart block.”

wherein C(X)BR2 can be CH2OH and R1 can be alkyl- or alkoxyalkyl. The compounds are disclosed to be useful as vasodilators or an antihypertensives.

Linden et al. (U.S. Pat. No. 5,877,180) is based on the discovery that certain inflammatory diseases, such as arthritis and asthma, may be effectively treated by the administration of compounds which are selective agonists of A2A adenosine receptors, preferably in combination with a Type IV phosphodiesterase inhibitor. An embodiment of the Linden et al. invention provides a method for treating inflammatory diseases by administering an effective amount of an A2A adenosine receptor of the following formula:

wherein R and X are as described in the patent.

In a preferred embodiment, the Linden et al. invention involves the administration of a Type IV phosphodiesterase (PDE) inhibitor in combination with the A2A adenosine receptor agonist. The Type IV phosphodiesterase (PDE) inhibitor includes racemic and optically active 4-(polyalkoxyphenyl)-2-pyrrolidones of the following formula:

wherein R′, R18, R19 and X are as disclosed and described in U.S. Pat. No. 4,193,926. Rolipram is an example of a suitable Type IV PDE inhibitor included within the above formula.

G. Cristalli (U.S. Pat. No. 5,593,975) discloses 2-arylethynyl, 2-cycloalkylethynyl or 2-hydroxyalkylethynyl derivatives, wherein the riboside residue is substituted by carboxy amino, or substituted carboxy amino (R3HNC(O)—). 2-Alkynylpurine derivatives have been disclosed in Miyasaka et al. (U.S. Pat. No. 4,956,345), wherein the 2-alkynyl group is substituted with (C3–C16)alkyl. The '975 compounds are disclosed to be vasodilators and to inhibit platelet aggregation, and thus to be useful as anti-ischemic, anti-atherosclerosis and anti-hypertensive agents.

However, a continuing need exists for selective A2 adenosine receptor agonists useful for therapeutic applications, that have reduced side effects.

SUMMARY OF THE INVENTION

The present invention comprises compounds and methods of their use for the treatment of inflammatory activity in mammalian tissue. The inflammatory tissue activity can be due to pathological agents or can be due to physical, chemical or thermal trauma, or the trauma of medical procedures, such as organ, tissue or cell transplantation, angioplasty (PCTA), inflammation following ischemia/reperfusion, or grafting. The present compounds comprise a novel class of 2-alkynyladenosine derivatives, substituted at the ethyne position by substituted cycloalkyl moieties. Preferably, the riboside residue is substituted at the 5′-position (“X”) by an N-alkyl-(or cycloalkyl)carboxyamino (“aminocarbonyl”) moiety. Thus, the present invention provides a method for inhibiting the inflammatory response in a mammal, such as a human subject, and protecting the tissue subject to the response, by administering an effective amount of one or more compounds of the invention.

The compounds of the invention have the following general formula (I):

(d) R1 is (X-(Z)-)n[(C3–C10)cycloalkyl]-(Z′)- wherein Z and Z′ are individually (C1–C6)alkyl, optionally interrupted by 1–3 S or non-peroxide O, or is absent, and n is 1–3; or a pharmaceutically acceptable salt thereof.

The invention provides a compound of formula I for use in medical therapy, preferably for use in treating or protecting tissue from inflammation such as an inflammatory response, as well as the use of a compound of formula I for the manufacture of a medicament for the treatment of an inflammatory response due to a pathological condition or symptom in a mammal, such as a human, which is associated with inflammation.

Although certain A2A adenosine receptor agonists have been reported to be vasodilators, and thus to be useful to directly treat hypertension, thrombus, atherosclerosis and the like, the tissue-protective activity of the compounds of formula (I) is not suggested by the prior art.

The invention also includes the use of a combination of these compounds with type IV phosphodiesterase inhibitors for synergistic decreases in the inflammatory response of immune cells.

The invention also provides a pharmaceutical composition comprising an effective amount of the compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable diluent or carrier, and optionally, in combination with a Type IV phosphodiesterase (PDE) inhibitor. Preferably, the composition is presented as a unit dosage form.

Additionally, the invention provides a therapeutic method for preventing or treating a pathological condition or symptom in a mammal, such as a human, wherein the activity of A2A adenosine receptors is implicated and agonism of said activity is desired, comprising administering to a mammal in need of such therapy, an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof. It is believed that activation of A2A adenosine receptors inhibits inflammation by effecting neutrophils, mast cells, monocytes/macrophages, T-cells and/or eosinophils. Inhibition of these inflammatory cells results in tissue protection following tissue insults.

Among the inflammatory responses that can be treated (including treated prophylactically) with a compound of formula I, optionally with a Type IV PDE inhibitor, are inflammation due to

(k) chemical or thermal trauma due to bums, acid, alkali and the like.

Of particular interest and efficacy is the use of the present compounds to treat inflammatory responses due to organ, tissue or cell transplantation, i.e., the transplantation of allogeneic or xenogeneic tissue into a mammalian recipient, autoimmune diseases and inflammatory conditions due to circulatory pathologies and the treatment thereof, including angioplasty, stent placement, shunt placement or grafting. Unexpectedly, it was found that administration of one or more compounds of formula (I) was effective after the onset of the inflammatory response, e.g., after the subject was afflicted with the pathology or trauma that initiates the inflammatory response.

The invention also includes a method for measuring the response, or binding a compound of formula I at or to designated A2A adenosine receptor sites comprising said receptors, in vivo or in vitro, with an amount of a compound of formula I effective to bind to said receptors. Tissue or cells comprising ligand bound receptor sites can be used to measure the selectively of test compounds for specific receptor subtypes, the amount of bioactive compound in blood or other physiological fluids, or can be used as a tool to identify potential therapeutic agents for the treatment of diseases or conditions associated with receptor site activation, by contacting said agents with said ligand-receptor complexes, and measuring the extent of displacement of the ligand and/or binding of the agent, or the cellular response to said agent (e.g., cAMP accumulation).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting the competition by compounds of the invention for binding to recombinant human A2A adenosine receptors.

FIG. 2 is a graph depicting the decrease in human neutrophil oxidative activity by compounds of the invention.

FIG. 3 is a graph depicting the inhibition of human neutrophil oxidative activity by a compound of the invention (JMR193) with and without rolipram.

FIG. 5 is a graph depicting the effect of a compound of the invention on neutrophil cAMP content and adherence to a biological surface.

FIG. 6 is a graph depicting the ability of compound DWH-146e to inhibit PMN superoxide release on a biological surface.

FIG. 7 is a graph depicting the ability of compound DWH-146e to reduce plasma creatinine following ischemia/reperfusion injury in rats.

FIG. 8 is a graph depicting the effect of compound ZM241385 on renal function that had been improved following I/R injury by DWH-146e.

FIG. 9 is a comparative image of treated and untreated sections of rat kidney subjected to I/R injury.

FIG. 10 is a graph depicting the effect of DWH-146e on arterial pO2 following lung reperfusion injury in rabbits.

FIG. 11 is a graph depicting the effect of DWH-146e on pulmonary muscular resistance following lung reperfusion injury.

FIG. 12 is a graph depicting the effect of DWH-146e on mycloperoxidase activity following lung reperfusion injury.

FIG. 13 is a graph depicting the effect of DWH-146e on neointimal formation after arterial injury in mice.

FIG. 14 is a graph depicting the effect of compounds JMR193, DWH-146e, and CGS21680 on TNF release from human monocytes.

FIG. 15 is a graph depicting the effect of compound ZM241385 on the effects of JMR193 on TNF production.

FIG. 16 is a graph depicting the activity of DWH-146e on mean leukocyte concentration (WBC/mm2) in a murine peritonitis model, following injection of zymosan (Zym).

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used, unless otherwise described. Halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, aralkyl, alkylaryl, etc. denote both straight and branched alkyl groups; but reference to an individual radical such as “propyl” embraces only the straight chain radical, a branched chain isomer such as “isopropyl” being specifically referred to. Aryl includes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Heteroaryl encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (C1–C4)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.

It will be appreciated by those skilled in the art that the compounds of formula (I) have more than one chiral center and may be isolated in optically active and racemic forms. Preferably, the riboside moiety of formula (I) is derived from D-ribose, i.e., the 3′,4′-hydroxyl groups are alpha to the sugar ring and the 2′ and 5′ groups is beta (3R, 4S, 2R, 5S). When the two groups on the cyclohexyl group are in the 4-position, they are preferably trans. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, or enzymatic techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine adenosine agonist activity using the tests described herein, or using other similar tests which are well known in the art.

Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.

A specific value for R1 is carboxy- or (C1–C4)alkoxycarbonyl-cyclohexyl(C1–C4)alkyl.

A specific value for R2 is H or (C1–C4)alkyl, i.e., methyl or ethyl.

A specific value for R3 is H, methyl or phenyl.

A specific value for R4 is H, methyl or phenyl.

A specific value for Z is —CH2— or —CH2—CH2—.

A specific value for X is CO2R2, (C2–C5)alkanoylmethyl or amido.

A specific value for n is 1.

Preferred compounds of formula (I) are those wherein each R is H, X is ethylaminocarbonyl and R1 is 4-carboxycyclohexylmethyl (DWH-146a), R1 is 4-methoxycarbonylcyclohexylmethyl (DWH-146e) or R1 is 4-acetoxymethyl-cyclohexylmethyl (JMR-193). They are depicted below (DWH-146 (acid) and methylester (e)) and JMR-193.

The synthesis of methyl 4[3-(6-amino-9(5-[(ethylamino)carbonyl]-3,4-dihydroxytetrahydro-Z-furanyl-9H-2-purinyl)-2-propynyl]-1-cyclohexane-carboxylate (DWH-146e) was accomplished by the cross coupling of an iodo-adenosine derivative (N-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-β-D-ribofuranuoramide) with methyl 4-(2-propynyl)-1-cyclohexanecarboxylate by utilization of a Pd11 catalyst. The synthesis of the iodo-adenosine derivative was accomplished from guanosine. Guanosine is first treated with acetic anhydride, which acetalates the sugar hydroxyls, followed by the chlorination of position 6 with tetramethyl ammonium chloride and phosphorousoxychloride. Iodination of position 2 was accomplished via a modified Sandmeyer reaction, followed by displacement of the 6-Cl and sugar acetates with ammonia. The 2′ and 3′ hydroxyls were protected as the acetonide and the 5′ hydroxyl was iodized to the acid with potassium permanganate. Deprotection of the 2′ and 3′ acetonide, Fisher esterification of the 5′ acid with ethanol and conversion of the resulting ethyl ester to the ethyl amide with ethylamine gave N-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-β-D-ribofuranuoramide.

The acetylene (methyl 4-(2-propynyl)-1-cyclohexanecarboxylate) was synthesized starting from trans-1,4-cyclohexanedimethanol. Initially the trans-diol was monotosylated followed by displacement of the tosylate with an acetylene anion. The hydroxyl of the resulting hydroxyl acetylene species was oxidized to the acid via Jones reagent followed by methylation with (trimethylsilyl)diazomethane to give methyl 4-(2-propynyl)-1-cyclohexanecarboxylate.

DWH-146e and JMR193 are substantially more potent as inhibitors in inflammatory model systems than the reference compound, CGS21680 (2-[p-(carboxyethyl)-phenyl-ethylamino]5′-N-ethylcarboxamidoadenosine). For example, DWH-146e is about 80 times more potent at A2A receptors and 40 times more selective for A2A over A3 receptors than is CGS21680.

Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

The compounds of formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949. Useful dosages of Type IV PDE inhibitors are known to the art. For example, see, U.S. Pat. No. 5,877,180, Col. 12.

Generally, the concentration of the compound(s) of formula (I) in a liquid composition, such as a lotion, will be from about 0.1–25% wt-%, preferably from about 0.5–10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1–5 wt-%, preferably about 0.5–2.5 wt-%.

The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about 0.5 to about 100 μg/kg, e.g., from about 10 to about 75 μg/kg of body weight per day, such as 3 to about 50 μg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 μg/kg/day, most preferably in the range of 15 to 60 μg/kg/day.

Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.1 to about 10 nM, preferably, about 0.2 to 10 nM, most preferably, about 0.5 to about 5 nM. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1–100 μg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.01–5.0 μg/kg/hr or by intermittent infusions containing about 0.4–15 μg/kg of the active ingredient(s).

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye. For example, it is desirable to administer the present compositions intravenously over an extended period of time following the insult that gives rise to inflammation.

The ability of a given compound of the invention to act as an A2A adenosine receptor agonist (or antagonist) may be determined using pharmacological models which are well known to the art, or using tests described below.

The invention will be further described by reference to the following detailed examples, which are given for illustration of the invention, and are not intended to be limiting thereof.

[(2R,3R,4R,5R)-3,4-diacetyloxy-5-(2-amino-6-chloropurin-9-yl)oxolan-2-yl]methyl acetate (6.3). To a 1000 mL flask was added 80 g (0.195 mol) [(2R,3R,4R,5R)-3-4-diacetyloxy-5-(2-amino-6-oxohyropurin-9-yl)oxolan-2-yl]methyl acetate (6.2), tetramethylammonium chloride (44 g, 0.4 mol), anhydrous acetonitrile (400 mL) and N,N-dimethylaniline (25 mL). The flask was placed in an ice salt bath and cooled to 2° C. To this solution was added dropwise POCl3 (107 mL 1.15 mol) at a rate that maintained the temperature below 5° C. (45 minutes). The flask was then removed from the ice bath, outfitted with a condenser, placed in an oil bath and allowed to reflux for 10 minutes whereas the solution changed to a red/brown color. The solvent was then removed under reduced pressure to yield an oily residue which was transferred to a beaker containing 1000 g of ice and 400 mL of CHCl3 and allowed to stir for 1.5 hours to decompose any remaining POCl3. The organic phase was then removed and the aqueous phase extracted with 3×50 mL of CHCl3 and pooled with the organic phase. The pooled organic was then back extracted with 50 mL of water followed by stirring with 200 mL of saturated NaHCO3. The organic was further extracted with NaHCO3 until the aqueous extract was neutral (2×). The organic was finally extracted with brine and then dried over MgSO4 for 16 hours. To the solution was added 800 mL of 2-propanol after which the solution was concentrated under reduced pressure. To the oily solid was added 200 mL of 2-propanol and the solution was refrigerated overnight. The crystalline product was filtered, washed, and allowed to dry overnight to give 6.3 (77%). 1H NMR (300 MHz, CD3OD) δ 8.31 (s, 1H, H-8), 7.00 (s, 2H, NH2) 6.06 (d, J=5.8 Hz, 1H, H-1′), 5.83 (t, J=6.16 Hz, 1H, H-2′), 5.67 (m, 1H, H-3′), 4.29 (m, 3H, H-4′,5′), 2.07 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.98 (s, 3H, Ac). 13C NMR (300 MHz, CD3OD) δ 171.0, 170.4, 170.2, 160.8, 154.6, 150.8, 142.2, 124.5, 85.8, 80.6, 72.8, 71.2, 63.9, 21.4, 21.3, 21.1.

(2S,1R,4R,5R)-4-(6-amino-2-iodopurin-9-yl)-7,7-dimethyl-3,6,8-trioxabicyclo[3.3.0]octane-2-carboxylic acid (6.7). To a stirred solution of 1.6 g (3.7 mmol) of [(1R,2R,4R,5R)-4-(6-amino-2-iodopurin-9-yl)-7-7-dimethyl-3,6,8-trioxabicyclo[3.3.0]oct-2-yl]methan-1-ol (6.6) in 200 mL of H2O was added 0.60 g of KOH and, dropwise, a solution of 1.70 g (10.8 mmol) of KMnO4 in 50 mL of H2O. The mixture was set aside in the dark at room temperature for 225 hours. The reaction mixture was then cooled to 5–10° C. and decolorized by a solution of 4 mL of 30% H2O2 in 16 mL of water, while the temperature was maintained under 10° C. using an ice-salt bath. The mixture was filtered through Celite and the filtrate was concentrated under reduced pressure to about 10 mL and then acidified to pH 4 with 2N HCl. The resulting precipitate was filtered off and washed with ether to yield 6.7 (70%) after drying as a white solid, m.p. 187–190° C. 1H NMR (300 MHz, DMSO-d6) δ 8.11 (s, 1H, H-8), 7.62 (s, 2H, NH2), 7.46 (s, 1H, COOH), 6.22 (s, 1H, H-1′), 5.42 (d, J=5.71 Hz, 1H, H-2′), 5.34 (d, J=6.16 Hz, 1H, H-3′), 4.63 (s, 1H, H-4′), 1.46 and 1.30 (s, 3H, C(CH3)2).

Methyl-4-(3-{9-[(4S,5S,2R,3R)-5-(N-ethylcarbamoyl)-3,4-dihydroxyoxolan-2-yl-6-aminopurin-2-yl)}prop-2-ynyl)cyclohexane-carboxylate (DWH-146e). To a degassed solution of 25 mg (0.063 mmol) of [(2S,3 S,4R,5R)-5-(6-amino-2-iodopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]-N-ethylcarboxamide (6.9), 16.9 mg (0.094 mmol) (5.5), and 0.75 mg CuI in 5 mL each of TEA and acetonitrile was added 15 mg of Pd(PPh3)4. The solution was stirred for 24 hours at 70° C. after which time the solution was filtered through celite and chromatographed on silica gel with MeOH-CHCl3 (5:95) to give DWH-146e (24%).

EXAMPLE 14

(4-prop-2-ynylcyclohexyl)methyl acetate (5.6). Acetic anhydride (0.92 mL, 8.25 mmol) and pyridine (0.2 mL, 2.5 mmol) were added to a solution of 5.3 (250 mg, 1.65 mmol) in 25 mL ether. The reaction was allowed to stir at ambient temperature for 24 hours. Water was added to the reaction and the organic was further extracted with 10% NaHCO3. The organic layer was dried with MgSO4 and evaporated. The residue was chromatographed on silica gel with EtOAc-Hexanes (5:95) to yield 5.6 (47%).

EXAMPLE 15

[4-(3-{9-(4S,5S,2R,3R)-5-(N-ethylcarbamoyl)-3,4-dihydroxyoxolan-2-yl]-6-aminopurin-2-yl}prop-2-ynyl)cyclohexyl]methyl acetate (JMR193). To a degassed solution of 125 mg (0.29 mmol) of [(2S,3S,4R,5R)-5-(6-amino-2-iodopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]-N-ethylcarboxamide (6.9), 150 mg (0.77 mmol) (5.6), and 1.0 mg CuI in 1.3 mL of TEA and 4 mL DMF was added to 25 mg of Pd(PPh3)4. The solution was stirred for 72 hours at 60° C. after which time the solution was filtered through celite and chromatographed on silica gel with MeOH-CHCl3 (5:95) to give JMR193 (10%).

EXAMPLE 16

Radioligand Binding Studies. Binding to A2A receptors was evaluated with the radioligand 125I-ZM241385. FIG. 1 depicts the competition by selective agonists for binding to recombinant human A2A adenosine receptors. DWH-146e is highly selective for the recombinant human A2A (hA2A) subtype. Selectivity for the A3 receptor (not shown) is less impressive, but still about 50-fold. DWH-146e is about 5 and 50 times more potent than WRC0470 and CGS21680, respectively (FIG. 1). An unexpected and interesting finding is that the ester, DWH-146e also is about 50 times more potent than the acid, DWH-146a (FIG. 1).

EXAMPLE 17Effect of DWH-146e and JMR193 on Neutrophil Oxidative Activity

Luminol-enhanced chemiluminescence, a measure of neutrophil oxidative activity, is dependent upon both superoxide production and mobilization of the lysosomal granule enzyme myeloperoxidase. The light is emitted from unstable high-energy oxygen species generated by activated neutrophils. Purified neutrophils (5–10×105/ml) were incubated in Hanks balanced salt solution containing 0.1% human serum albumin (1 ml) with or without DWH-146a, DWH-146e, CGS21680, or JMR193 with or without rolipram and with or without tumor necrosis factor-alpha (1 U/ml) for 30 minutes at 37° C. in a shaking water bath. Then luminol (1×10−4 M) enhanced f-met-leu-phe (1 mcM) stimulated chemiluminescence was read with a Chronolog® Photometer (Crono-log Corp., Havertown, Pa.) at 37° C. for 2–4 minutes. Chemiluminescence is reported as relative peak light emitted (=height of the curve) compared to samples with tumor necrosis factor-alpha and without DWH, JMR or rolipram.

D. Results

As shown in FIG. 2, JMR193 and DWH-146e both decreased tumor necrosis factor-alpha-primed f-met-leu-phe-stimulated human neutrophil oxidative activity as measured by luminol-enhanced chemiluminescence more effectively than the adenosine A2A receptor agonist CGS21680. The horizonal axis gives the concentration of CGS21680, DWH-146a, DWH-146e or JMR193 (log nM). The vertical axis gives the resulting peak human neutrophil activity as relative amount of stimulated release of reactive oxygen species as measured with luminol-enhanced chemiluminescence compared to control samples which were not primed with tumor necrosis factor-alpha. Means SEM (n=4–5 separate experiments).

As shown in FIG. 6, inhibition of tumor necrosis factor-alpha (TNF)-stimulated adherent human neutrophil superoxide release on a fibrinogen-coated surface was accomplished by rolipram (300 nM) and DWH-146e. DWH-146e decreased the oxidative burst of adhering neutrophils, and synergistically decreased the oxidative burst in the presence of rolipram, which by itself did not affect neutrophil oxidative activity. The horizontal axis gives the DWH-146e concentration in nM and the vertical axis gives the amount of superoxide released by the neutrophils as measured by cytochrome c reduction. There was marked synergy with DWH-146e and the type IV PDE inhibitor, rolipram, to decrease tumor necrosis factor-alpha-stimulated adherent human neutrophil oxidative activity. Means SEM of replicates from 4–5 separate experiments. *p<0.05 decreased superoxide release compared to without DWH-146e; **p<0.05 decreased superoxide release compared to with rolipram and without DWH-146e.

EXAMPLE 18Treatment of Ischemia/Reperfusion (I/R) Injury in Kidney with DWH-146e

To determine whether or not the effect of DWH-146e on reduction of plasma creatinine in rats subjected to I/R is A2A-receptor mediated, rat kidneys were subjected to 45 minutes ischemia followed by 48 hours reperfusion. DWH-146e (0.004 μg/kg/min) was administered continuously via minipump beginning 5 hours prior ischemia. As shown in FIG. 8, the improvement in renal function was reversed by the A2A antagonist ZM-241385 (0.003 μg/kg/min-equimolar delivery rate compared with DWH-146e) (*P<0.001 for Vehicle vs. DWH; **P<0.05 DWH vs. DWH/ZM. N=5 for Vehicle, DW; N=6 for DWH/ZM. ANOVA followed by Bonferroni correction).

DWH-146e, at concentrations that have no hemodynamic effects, prevents renal edema, necrosis and red cell pooling in the inner medulla.

The protection against renal damage afforded by DWH-146e (0.01 μg/kg/min s.c. for 48 hours) was correlated with a dramatic inhibition of neutrophil adherence to vascular endothelium. It is believed that inhibition by DWH-146e of the interaction between neutrophils and vascular endothelium is responsible, at least in part, for the protection against renal damage.

To determine whether or not A2A-AR activation reduces neutrophils in the outer medulla of rats subjected to I/R, using Neurolucida®, the kidney was viewed under 100× mag and the entire kidney was drawn. PMNs were counted by viewing kidney sections under 250× mag. Kidney sections were overlaid with optical frames viewed under the microscope and all PMNs were counted within each frame. This system prevents counting of PMNs more than once. As shown in FIG. 9, the density of neutrophils was 15.65/mm2 for vehicle and 3.02/mm2 for DWH-146e treatment.

EXAMPLE 19Effect of DWH-146e on Lung Reperfusion Injury

A. Methods. An isolated, whole blood-perfused, ventilated rabbit lung model was used. Donor rabbits underwent lung harvest after pulmonary arterial PGE1 injection and Euro-Collins preservation solution flush, and lungs were preserved for 18 hours at 4° C. Group I lungs (n=9) served as control subjects. Group II lungs (n=9) were reperfused with whole blood that was first passed through a leukocyte-depleting filter. In group III (n=9), DWH-146e was added to the blood reperfusate (25 μg/kg) immediately before reperfusion and was administered throughout the reperfusion period (1 μg/kg/min). All lungs were reperfused for 30 minutes, and pulmonary artery pressure (PAP), pulmonary vascular resistance (PVR), airway compliance (CPL) and arterial oxygenation were recorded. Mycloperoxidase activity (MPO) was recorded to quantify neutrophil sequestration, and wet/dry weight ratios were measured to demonstrate pulmonary edema.

B. Results. Arterial oxygentation in group II and group III was significantly higher than that of group I after 30 minutes of reperfusion (514.27±35.80 and 461.12±43.77 vs. 91.41±20.58 mm Hg, p<0.001. As shown in FIG. 10, group III lungs displayed a progressive involvement in pO2 throughout reperfusion. Leukocyte depletion in group II lungs improved arterial oxygenation in early reperfusion. *p=0.004 (group II versus groups I and III); **p<0.001 (groups II and III versus group I).

As shown in FIG. 11, mean PVR in group II was significantly reduced when compared to controlled lungs (*p<0.001). PVR of group III lungs was significantly lower than even those lungs that underwent reperfusion with leukocyte-depleted blood (**p<0.001 versus groups I and II). Pulmonary vascular resistance was significantly reduced in group III (22,783±357 dynes·s·cm−5) compared to both group II and group I (31,057±1743 and 36,911±2173 dynes·s·cm−5, p<0.001). Airway compliance was improved in groups II and III when compared to group I (1.68±0.08 and 1.68±0.05 vs. 1.36±0.13, p=0.03). Microvascular permeability in group III was reduced to 106.82±17.09 compared with 165.70±21.83 ng Evans-blue dye/gm tissue in group I (p=0.05). As shown in FIG. 12, myeloperoxidase activity in group III was significantly lower than in group I (*p=0.03). MPO=myeloperoxidase. Group III myeloperoxidase activity was 39.88±4.87 compared with 88.70±18.69 ΔOD/gm/min in group I (p=0.03), and group II myeloperoxidase activity was 56.06±7.46.

C. Conclusions. DWH-146e reduced lung neutrophil sequestration and dramatically improved pulmonary graft function. Neutrophils are important components of the inflammatory cascade of reperfusion injury and their source may include both the circulating blood and the lung graft itself. Selective adenosine-A2A activation interrupts the neutrophil-mediated inflammatory response and reduces lung reperfusion injury following transplantation.

Under light microscopy, control lungs in group I showed severe leukocyte infiltration and edema formation in the alveolar spaces after 18 hours of ischemic storage and 30 minutes of reperfusion. In group II, lungs that underwent reperfusion with leukocyte-depleted blood and in group III lungs (that received DWH-146e during reperfusion, this infiltration was much less.

EXAMPLE 20

Effect of DWH-146e on Neointimal Formation after Arterial Injury. Leukocyte activation with release of inflammatory cytokines occurs after percutaneous coronary intervention and may play a role in restenosis. In the mouse, robust neointima formation in the presence of an intact endothelial lining occurs after ligation of the common carotid artery. Using this model, C57/BL6 mice were randomized at the time of carotid ligation to a 7 day infusion via osmotic pump of DWH-146e, (n=7), or vehicle (n=8).

At 14 days after carotid ligation, histomorphometry demonstrated a significant reduction in neointimal area (0.005±0.004 mm2 vs. 0.021±0.014 mm2, p=0.02) and neointimal to medial area ratio (0.13±0.07 vs. 0.64±0.44, p=0.01) in the treated animals compared to controls. Medial area was similar in the two groups (0.034±0.007 mm2 vs. 0.036±0.009 mm2, p=0.81). This benefit in limiting neointimal growth persisted to 28 days. FIG. 13 summarizes the effect of DWH-146e to inhibit neointimal growth in the mouse LCCA model. These experiments demonstrate that, in a mouse carotid artery ligation model, prolonged A2A stimulation (7 days) by DWH-146e results in a significant reduction in neointimal formation for at least 21 days, possibly through its effect on leukocyte activation and function.

Thus, DWH146e and JMR193 decrease LPS endotoxin-stimulated TNFα production by human monocytes by a mechanism that is dependent upon agonist binding to A2A adenosine receptors.

EXAMPLE 22Activity of DWH-146e in Murine Peritonitis Model

Preliminary experiments with experimental peritonitis have involved the injection of zymosan (Zym) as a potent stimulus of inflammation (Y. Zhang et al., Eur. J. Pharmacol., 313, 237 (1996)). As shown in FIG. 16, following injection of zymosan, the mean leukocyte concentration as determined in a neubauer hemocytometer was 7,325±1,893/mm3. Intraperitoneal injection of DWH-146e at a dosage of 2.5 μg/kg one hour prior to zymosan inhibited the development of peritonitis with a mean±SEM leukocyte concentration of 2,012±374/mm3 6 hours later (p<0.05). Thus, these studies demonstrate that the A2A AR is instrumental in mediating PMN traversal into the peritoneum following zymosan challenge.

All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.